HRP20040704A2 - A nutrient pharmaceutical formulation comprising polyphenols and use in treatment of cancer - Google Patents

Abstract

A nutrient pharmaceutical formulation composition useful for treating cancer comprising ascorbic acid, L-lysine, L-proline and at least one polyphenol compound selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, catechin and the use of the composition in treating cancer and other tumors are provided. A nutrient pharmaceutical formulation composition of EPICAN FORTE(TM) and its method of use in treating cancer are also disclosed.

Description

Reference to related application

This application claims benefit under 35 U.S.C. § 119(e) U.S.C. provisional application serial number 60/348,143 filed on Jan. 11, 2002, the contents of which are incorporated herein by reference in their entirety.

Field of invention

The present invention relates to a nutritional pharmaceutical formulation and its use for the treatment of cancer. In particular, the subject invention relates to pharmaceutical formulations that contain polyphenols and have an effective amount of polyphenols for the treatment of cancer. The formulation of the present invention is used as an agent for the prevention and treatment of cancer, containing ascorbic acid, lysine, proline and at least one polyphenolic compound selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin and catechin.

State of the art

Cancer is one of the leading causes of death in the industrialized world. There is no specific and causative treatment for cancer, and cancer mortality is among the highest of all diseases. The most widely used treatments, chemotherapy and radiotherapy, do not distinguish between healthy tissue and cancer and are associated with severe side effects. This is why there is a need for selective cancer treatment.

One of the key mechanisms used by cancer cells to expand and metastasize in the body involves the enzymatic destruction of surrounding connective tissue. Therapeutic approaches to control this process through specific drugs have not been successful and currently there are no available means to control cancer metastases. Current treatment protocols with chemotherapy and radiotherapy focus on the destruction of cancer cells in the body, and they are not aimed at metastasizing. Moreover, these treatments are toxic, non-specific and associated with severe side effects. Chemotherapy and radiotherapy carry the risk of developing new cancers, and by destroying the connective tissue in the body, they can facilitate the invasion of cancer.

In order to grow and expand to other parts of the body, cancer cells destroy the extracellular matrix through various matrix metalloproteinases (Matrix Metalloproteinases, MMPs) and plasmin, whose activities have been correlated with the aggressiveness of tumor growth. Rath and Pauling (1992) hypothesized that nutrients such as some amino acids, lysine and ascorbic acid, may act as natural inhibitors of extracellular matrix proteolysis and as such have the potential to modulate tumor growth and expansion. These nutrients may exert their antitumor potential through several mechanisms, among them inhibition of MMPs and strengthening of the connective tissue surrounding cancer cells (tumor "encapsulation" effect).

LOUSE. patent no. 5,962,517 discloses a pharmaceutical composition for a different medical indication (treatment of acne). The disclosed composition comprises an acne-reducing component, at least one of burdock root and sorrel root, or a catechin-based composition; and a skin cell conditioning component containing a transition metal. The disclosed composition is not shown to have any beneficial value in the treatment and/or prevention of cancer.

PCT WO 00/76492 discloses a disease-reducing nutritional formulation containing a catechin compound. However, the bioavailability of catechin compounds has been shown to be low (Chen L., Lee M.J., Yang C.S. Drug. Metab. Dispos. 25: 1045-1050 (1997); Yang C.S., Chen L., Lee M.J.., Balentine D.A., Kuo M.C., Schantz S. Cancer Epidemol. Biomark. Prev. 7: 351-35 (1998); Bell J.R., Donovan J.L., Wong R., Waterhouse H., German J.B., Walzem R.L., Kasim K. Am. J. Clin. Nutr. 71: 103-108 (2000); Sherry Chow H.H., Cai Y., Alberts D.S., Hakim I., Dorr R., Shahi F., Crowell J.A., Yang S.C., Hara H. Cancer Epidemol. Biomark. Prev. 10: 53-58 (2001)), and PCT WO 00/76492 fails to disclose a way to increase bioavailability as needed in the treatment and/or prevention of cancer.

Demeule et al. reveal that green tea catechins can have inhibitory effects on matrix metalloproteinase. There are no recommendations or interpretations regarding how to use catechins in the treatment and/or prevention of cancer. Considering the fact that the bioavailability of polyphenols in humans is extremely low, the low tissue concentration of catechins greatly reduces the therapeutic value of polyphenols including epigallocatechin gallate (EGCG). There is a constant need to find a better pharmaceutical composition containing polyphenols that is effective in the treatment of neoplastic diseases and other diseases including inflammatory diseases.

There is a need for safe and effective natural approaches that can be used to control the process of cancer expansion in the body. There is also a need for a preventive measure against cancers or benign tumors developing in humans and such a measure could be applied to patients without the risk of side effects associated with treatment. Nutritive pharmaceutical formulations have become a favorite, as the incidence of cancer has recently increased. The demand for such composition will continue and is likely to grow.

Presentation of the essence of the invention

One object is that the subject invention provides a nutritional pharmaceutical composition useful in the treatment of cancer, which contains: a) one ascorbic compound; b) one L-lysine compound; c) one L-proline compound; and d) at least one polyphenolic compound selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin and catechin. Compounds from a) to c) enhance the action of the polyphenolic compound in blocking the proliferation and metastasis of cancer.

Suitably, the ascorbic compound is selected from the group consisting of ascorbic acid, pharmaceutically acceptable ascorbate salts, ascorbate esters and/or mixtures thereof. Conveniently, the pharmaceutically acceptable ascorbate salt is calcium ascorbate or magnesium ascorbate. More preferably, the ascorbate ester is ascorbyl palmitate. Suitably, the lysine compound is selected from the group consisting of lysine hydrochloride and pharmaceutically acceptable salts of lysine. Suitably, the proline compound is selected from the group consisting of proline hydrochloride and pharmaceutically acceptable proline salts.

Another goal is that the nutritional pharmaceutical composition in question further contains some trace element selected from the group consisting of selenium, copper, manganese, calcium and magnesium.

Another goal is that the nutritional pharmaceutical composition in question further contains an amino acid. Conveniently, the amino acid is arginine. More preferably, the subject nutritional pharmaceutical composition further comprises N-acetyl cysteine.

Another goal is that the present invention provides a nutritional pharmaceutical composition useful in the treatment of cancer, which contains: a) one ascorbic compound; b) one L-lysine compound; c) one L-proline compound; d) N-acetyl cysteine; and e) at least one polyphenolic compound selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin and catechin. Compounds a) to d) enhance the action of the polyphenol compound in blocking the proliferation and metastasis of cancer.

Another goal is that the present invention provides a nutritional pharmaceutical composition containing 250 mg of ascorbic acid, 250 mg of calcium ascorbate, 250 mg of magnesium ascorbate, 250 mg of ascorbyl palmitate, 1000 mg of polyphenols, 200 mg of N-acetyl-cysteine, 1000 mg of lysine, 750 mg proline, 500 mg arginine, 30 mcg selenium, 2 mg copper and 1 mg manganese.

Another goal is that the present invention provides a nutritional pharmaceutical composition containing 25 mg of ascorbic acid, 25 mg of calcium ascorbate, 25 mg of magnesium ascorbate, 25 mg of ascorbyl palmitate, 200 mg of polyphenols, 10 mg of N-acetyl-cysteine, 50 mg of lysine, 25 mg proline, 50 mg arginine, 1 mcg selenium, 20 mcg copper and 50 mcg manganese.

Another goal is that the present invention provides a nutritional pharmaceutical composition containing 5000 mg of ascorbic acid, 5000 mg of calcium ascorbate, 5000 mg of magnesium ascorbate, 5000 mg of ascorbyl palmitate, 5000 mg of polyphenols, 1500 mg of N-acetyl-cysteine, 5000 mg of lysine, 3000 mg proline, 3000 mg arginine, 200 mcg selenium, 9 mg copper and 10 mg manganese.

Another goal is that the present invention provides a nutritional pharmaceutical composition containing 250 mg of ascorbic acid, 250 mg of calcium ascorbate, 250 mg of magnesium ascorbate, 250 mg of ascorbyl palmitate, 1000 mg of polyphenols, 200 mg of N-acetyl-cysteine, 1000 mg of lysine, 750 mg proline, 500 mg arginine, 100 mcg selenium, 2 mg copper, 1 mg manganese, 500 mg calcium and 400 mg magnesium.

Another goal is that the present invention provides a nutritional pharmaceutical composition containing 250 mg of ascorbic acid, 250 mg of calcium ascorbate, 250 mg of magnesium ascorbate, 250 mg of ascorbyl palmitate, 1000 mg of polyphenols, 200 mg of N-acetyl-cysteine, 1000 mg of lysine, 750 mg proline, 500 mg arginine, 100 mcg selenium, 2 mg copper, 1 mg manganese, 500 mg calcium, 400 mg magnesium and 200 mg citrus bioflavonoids.

Another goal is that the present invention provides a nutritional pharmaceutical composition useful in the treatment of cancer, which contains: L-lysine, L-proline, L-arginine, ascorbic acid, calcium, magnesium, polyphenols, N-acetyl-cysteine, selenium, copper and manganese.

Another goal is that the present invention provides a nutritional pharmaceutical composition containing 1000 mg of L-lysine, 750 mg of L-proline, 500 mg of L-arginine, 710 mg of ascorbic acid, 22 mg of calcium, 50 mg of magnesium, 1000 mg of polyphenols, 200 mg of N-acetyl-cysteine, 30 mcg of selenium, 2 mg of copper and 1 mg of manganese.

It is yet another object that the subject invention provides a method for treating cancer in an individual, comprising the step of administering to that individual a nutritional pharmaceutical composition as disclosed. Suitably, the cancer is selected from the group consisting of melanoma cancer, breast cancer, colon cancer, lung cancer and brain cancer.

Another object is that the subject invention provides a method for treating an inflammatory disease in an individual, which includes the step of administering to that individual a nutritional pharmaceutical composition as disclosed. Conveniently, the inflammatory disease is osteoarthritis.

Brief description of the drawing

Figure 1 shows the inhibitory effects of polyphenols, ascorbate, proline and lysine on the migratory behavior of human breast cancer cells.

Figure 2 shows the inhibitory effects of polyphenols, ascorbate, proline and lysine on the migration behavior of colon cancer cells in humans.

Figure 3 shows the inhibitory effects of polyphenols, ascorbate, proline and lysine on the migration behavior of human melanoma cells.

Figure 4 shows the inhibitory effects of polyphenols, ascorbate, proline and lysine on the apoptosis of human melanoma cells.

Figure 5 shows the inhibitory effects of ascorbic acid, proline, lysine and different amounts of EGCG on cell proliferation of human melanoma cells (A 2058).

Figure 6 shows the inhibitory effects of EGCG on cell proliferation of human breast cancer cells (MDA-MB 231).

Figure 7 shows the effects of supplementation (enrichment) of the basal medium with ascorbic acid, proline, lysine and different amounts of EGCG on cell proliferation of human breast cancer cells (MDA-MB 231).

Figure 8 shows the effects of supplementing the basal medium with ascorbic acid, proline, lysine and different amounts of EGCG on cell proliferation of human colon cancer cells (HCT 116).

Figure 9 shows the effects of supplementing the basal medium with ascorbic acid, proline, lysine and different amounts of EGCG on the expression of matrix metalloproteinases (MMPs) in human melanoma cells.

Figure 10 shows the effects of supplementing the basal medium with ascorbic acid, proline, lysine and different amounts of EGCG on Matrigel invasion by human breast cancer cells (MDA-MB 231).

Figure 11 shows the effects of supplementing the basal medium with ascorbic acid, proline, lysine and different amounts of EGCG on Matrigel invasion by human colon cancer cells (HTC 116).

Figure 12 shows the effects of supplementing the basal medium with ascorbic acid, proline, lysine and different amounts of EGCG on reducing the number of invasive cells in human melanoma cells (A 2058).

Detailed description of the invention

As used herein, "lysine" is used interchangeably with L-lysine, "proline" is used interchangeably with L-proline, "arginine" is used interchangeably with "L-arginine"; "vitamin C" is used interchangeably with ascorbic acid and may include ascorbate salts or ascorbate esters. "MMPs" refer to matrix metalloproteinases; for example MMP-1, MMP-2, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, etc. "EGCG" refers to (-)-epigallocatechin-3-gallate which is the main polyphenolic ingredient present in green tea. "NHDF" refers to normal human dermal fibroblast (Normal Human Dermal Fibroblast), including human chondrocytes and human stromal cells.

The present invention provides a nutritional pharmaceutical composition containing ascorbic acid, lysine, proline and at least one polyphenolic compound. Suitably, the ascorbic compound is selected from the group consisting of ascorbic acid, ascorbate salts and ascorbate esters. Suitably, the lysine is lysine hydrochloride or pharmaceutically acceptable salts of lysine. Suitably, the proline is proline hydrochloride or pharmaceutically acceptable proline salts thereof.

Polyphenolic compounds are green tea extracts. They are also known as catechins. Polyphenols are present in green tea and have been hypothesized to confer protection against various diseases including cancer (Mukhtar H., Ahmed N. Am. J. Clin. Nutr. 71:1698S-1702S (2000). Oral administration of green tea enhances the effects inhibition of tumors caused by doxorubicin in mice. The potential anticancer activity of catechins may be related to their effects on several factors involved in the proliferation of cancer cells and their metastasis. Catechins are known to cause cell cycle arrest in human cancer cells (Ahmad N., Feyes D.K., Nieminen A.L., Agarwal R., Mukhtar H.J. Natl. Cancer. Inst. 89: 1881-1886 (1997). A polyphenolic fraction from green tea has also been implicated in protecting against tumor-induced inflammation and cytokines.

Polyphenolic compounds represent 30% of the dry weight of green tea. They include flavanols, flavanediols, flavonoids and phenolic acids. Flavanols are the most abundant polyphenols in green tea and are commonly known as catechins. There are four main catechins in green tea: 1) (-)-epicatechin, 2) (-)-epicatechin-3-gallate, 3) (-)-epigallocatechin, and 4) (-)-epigallocatechin-3-gallate (EGCG). . Among the catechins, EGCG is the main polyphenolic constituent present in green tea. EGCG is a potent antioxidant compound and may contribute to the anticancer effects of green tea. Catechin compounds have been reported to exert their antimetastatic effect by preventing the angiogenesis process (Cao Y., Cao R. Nature 398:381 (1999)). EGCG has also been shown to interfere with the action of urokinase (u-plasminogen activator) (Jankun J., Selman S.H., Swiercz R., Skrzypczak J.E. Nature: 387-567 (1997), one of the most commonly expressed enzymes in human cancers. EGCG is suitable polyphenol compound.

Suitably, the polyphenolic compound is selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, catechin and other pharmaceutically acceptable polyphenolic salts and/or mixtures thereof.

One of the main therapeutic goals of this patent application is the prevention of digestion of the extracellular matrix and its restoration. Ascorbic acid or its salt (i.e. ascorbate) is necessary for the synthesis of collagen, elastin and other key molecules of the extracellular matrix.

Conveniently, a combination of ascorbic acid, proline and lysine is used to enhance the anticancer activity of the polyphenolic compounds. More preferably, the combination enhances polyphenolic compounds including epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin and catechin in such a way that the polyphenolic compound is effective in reducing cancer cell invasion or completely blocking invasion.

Without being limited by any theory, the subject nutritional pharmaceutical formulation contains a mixture of ingredients including polyphenols and this mixture has been found to function effectively in blocking the proliferation and metastasis process. We found that the subject composition of the nutritional formulation significantly reduces the invasion of cancer cells or completely blocks the metastasis of cancer cells. Therefore, this composition effectively prevented cancer cells from spreading. The therapeutic use of this patent-pending compound is an effective, selective and safe treatment for breast, colon and other organ cancers. Conveniently, the disclosed nutritional pharmaceutical composition may contain two of these components which are covalently linked.

Conveniently, the efficacy of the nutritional composition is enhanced by the inclusion of compounds known to beneficially affect the growth and invasion of cancer and other tumors, including L-arginine and/or arginine-containing compounds. More conveniently, N-acetyl-cysteine is used. More conveniently, some selenium salt, some copper salt and some manganese salt are used, which are effective in preventing and treating cancer and other tumors. This composition may also include one or more compounds required as a coenzyme in the Krebs cycle, respiratory chain, or other metabolic functions of cells in an amount effective to prevent and treat cancer and other tumors.

The nutritional pharmaceutical composition in question can be administered to the patient in the form of capsules, tablets, powders, pills, injections, infusions, inhalations, suppositories or other pharmaceutically acceptable carriers and/or means of delivery, for the prevention and treatment of cancer and other tumors. Conveniently, the nutritional pharmaceutical composition is administered as capsules, tablets or powders. More conveniently, it is given as capsules.

The nutritional pharmaceutical composition in question can be used in the prevention and treatment of cancer and other tumors in a selected patient, where the tumor or cancer is located in the breast, ovaries, cervix and other organs of the female reproductive system, lungs, liver, skin, gastrointestinal system, brain, bones or other body organs. Suitably, the composition is used for lung cancer and brain cancer.

The nutritional pharmaceutical composition in question can also be used in the prevention and treatment of infectious diseases, atherosclerosis, restenosis, other cardiovascular diseases and inflammatory diseases. Conveniently, the inflammatory disease is osteoarthritis.

MMP-mediated plasmin activation: It is speculated that MMP activity may be influenced by lysine through plasmin-mediated mechanisms, although other mechanisms have not been ruled out. MMPs are secreted as proenzymes and their activation is partially mediated by plasmin and its termination requires the active form of MMP-3. The mechanism of activation of various MMPs, described in detail by Nagase (1997), indicates that MMP-3 also requires the conversion of plasminogen to its active form, plasmin. The active center for plasminogen binding has sites where lysine specifically binds. Therefore, lysine can interfere with the activation of plasminogen into plasmin by plasminogen activator (Rath and Pauling, 1992), precisely by binding to the active sites of plasminogen. Tranexamic acid, a synthetic analog of lysine, has been used to inhibit plasmin-induced proteolysis through this mechanism.

Since the action of plasmin is essential for inducing several tissue MMPs, lysine can interfere with the conversion of plasminogen to plasmin and thus it can inhibit the activation of almost all MMPs. In addition, EGCG can exert an inhibitory effect on the degradation of the extracellular matrix by inhibiting MMP-2.

Cancer invasion and the role of the matrix: It is also possible to influence the invasion of cancer cells into the matrix by increasing the stability and strength of the connective tissue surrounding the cancer cells and contributing to the "encapsulation" of the tumor. This requires optimizing the synthesis and structure of collagen fibers, for which the hydroxylation of hydroxyproline and hydroxylysine residues in collagen fibers is essential. Ascorbic acid may be essential for the hydroxylation of these amino acids. Ascorbic acid and L-lysine are not normally produced in the human body; therefore, suboptimal levels of these nutrients are possible in different pathological stages as well as due to inappropriate diets. Although proline can be synthesized from arginine, its synthesis and/or hydroxylation may be affected under pathological conditions. As such, the hydroxyproline content of metastatic tumor tissue has been shown to be much lower than in non-metastatic tumor tissue (Chubinskaia et al, 1989). Various drugs that reduce metastasis also increased the hydroxyproline content of these tissues (Chubinskaia et al., 1989). Urinary hydroxyproline content in cancer patients was found to be higher than in healthy individuals or in non-cancer patients (Okazaki et al., 1992). All these findings suggest adverse effects of cancer cells on proline metabolism and possible conditional proline deficiency in cancer patients.

Cancer patients may have insufficient levels of ascorbic acid. Ascorbic acid can be cytotoxic to malignant cell lines and exert an antimetastatic effect. The present invention reveals that a combination of ascorbic acid, proline, lysine and at least one polyphenolic compound exerts a strong antiproliferative and antimetastatic effect on cancer cell lines. Suitably, the subject nutritive pharmaceutical formulation is effective against melanoma, breast cancer and colon cancer cells. Most preferably, the subject pharmaceutical formulation is effective against human colon cancer cells.

Polyphenolic compounds (catechins): EGCG is one of the catechins in green tea extract and may have growth inhibitory effects against human cancer cells.

The exact underlying mechanism is unclear. The present invention reveals a surprising synergy in a nutritional pharmaceutical formulation containing ascorbic acid, proline and lysine, and at least one polyphenolic compound in effectively blocking the proliferation and metastasis of cancer cells.

The nutritious pharmaceutical composition according to the subject invention effectively blocks the invasion of breast cancer, colon cancer, skin (melanoma) and other forms of cancer. The ingredients present in the nutritional formulation are naturally occurring compounds; and when used in the required range as disclosed in the application, they are shown to have no toxic side effects. In this way, this composition can also be used prophylactically, i.e. for effective prevention of cancer and other tumors in the body.

Since virus cells and other invasive microorganisms use similar proteases as cancer cells to spread infection in the body, this composition claimed in this patent can be used for effective prevention and treatment of viral diseases and other infectious diseases.

A similar mechanism of activation of matrix metalloproteinases, which cancer cells use to invade the extracellular matrix, also contributes to the destabilization of atherosclerotic plaques that lead to myocardial infarction and stroke. Therefore, this composition, for which protection is sought in this patent, can also be used for effective prevention and treatment of atherosclerosis, restenosis and other cardiovascular complications.

Activation of matrix metalloproteinases, which cancer cells use to invade the matrix, is a key component in various inflammatory conditions. Therefore, the composition claimed in this patent can also be used for the effective prevention and treatment of diseases such as rheumatoid arthritis, emphysema, allergies, osteoarthritis, and other conditions involving inflammatory aspects.

The nutritional pharmaceutical composition according to this invention can be administered to the patient in the form of tablets, pills, injections, infusions, inhalations, suppositories or other pharmaceutically acceptable carriers and/or methods of administration.

EXAMPLES

The effects of ascorbic acid, lysine, proline and at least one polyphenolic compound have been studied for their anti-proliferative and anti-invasive potential in various human cancer cell lines. One component of green tea extract (i.e. Epigallocatechin Gallate, EGCG) was investigated in particular.

Materials and procedures

Human breast cancer cells MDA-MB-231, human colon cancer cells HCT116, human melanoma cell line A2058 were obtained from ATCC. Normal human skin fibroblasts were obtained from GICBO. Where not noted, cell culture media supplied by ATCC were used.

In studies of cancer cell proliferation, each treatment was repeated eight times. In the invasion experiments, each treatment was performed three or four times.

Cell proliferation studies

In these studies, 5 × 104 breast cancer cells were grown in Liebovitz's medium with 10% fetal bovine serum (FBS) in 24-well plates. The medium was used as such (basal) or with indicated supplements (enrichment). The plates were incubated in an incubator with air circulation (without additional CO2) for four days. HCT 116 colon cancer cells were grown in McCoy's 5A medium and maintained in a 5% CO2 incubator with air circulation. At the end of the incubation period, the medium was removed and the cells in the wells were washed with PBS, followed by incubation with MTT dye for 3 hours. Dimethyl sulfoxide (DMSO, 1 ml) was added to each well. DMSO plates were allowed to stand at room temperature for 15 min with gentle shaking, and then the OD of the solution in each well was measured at 550 nm. The OD550 of the DMSO solution in the well was considered to be directly proportional to the number of cells. The OD550 of the treatment that did not contain any supplement (basal) was considered as 100.

Matrigel invasion studies

Studies were performed using Matrigel (Becton Dickinson) inserts in compatible 24-well plates. Fibroblasts were seeded and grown in wells of the plate using DMEM. When the fibroblasts reached confluence, the medium was withdrawn and replaced with 750 μl of the medium designated for processing. Cancer cells (5 x 104) suspended in 250 μl of nutrient-supplemented medium as specified in the experimental design were seeded onto the well insert. In this way, the medium on the insert and in the well contained the same supplements. Plates with inserts were then incubated (in an incubator with air circulation for MDA-MB-231 cells, and for colon cancer cells and melanoma cells in an incubator with 5% CO2) for 18-20 hours. After incubation, the media from the wells were withdrawn. The cells on the upper surface of the insert are slightly scraped with cotton wool on a stick. Those cells that penetrated the Matrigel membrane and migrated to the lower surface of the Matrigel were stained with hemacolor dye (EM Sciences) and visually counted under a microscope. Results were subjected to ANOVA and all possible pairs were tested for significance at p < 0.05.

Media in various studies were supplemented with ascorbic acid, proline, lysine, and EGCG at concentrations as indicated.

Gelatinase Zymography

Gelatinase zymography was performed in 10% Novex precast polyacrylamide gel (Invitrogen) in the presence of 0.1% gelatin. Culture media (20 μl) were applied and SDS-PAGE was performed with tris-glycine SDS buffer. After electrophoresis, gels were washed with 5% Triton X-100 for 30 min and stained. Protein standards were simultaneously separated and approximate molecular weights were determined.

Example 1

Breast cancer cells MDA-MB 231 (ATCC) were seeded on a Matrigel insert in a perfected Matrigel invasion chamber (BD). Conditioned media from normal human skin fibroblasts (Clonetics) supplemented with various agents (as indicated in Figure 1) were added to the well. The chamber was incubated for 24 hours and the cells, which invaded through the Matrigel membrane and migrated to the lower surface of the membrane, were counted. These data demonstrate the inhibitory effect of epigallocatechin gallate and a combination of ascorbic acid, proline and lysine on Matrigel invasion and migration of MDA-MB 231 human breast cancer cells.

Example 2

Human colon cancer cells HCT 116 (ATCC) were seeded on a Matrigel insert in a perfected Matrigel invasion chamber (BD). Conditioned media from normal human skin fibroblasts (Clonetics) supplemented with various agents (as indicated in Figure 2) were added to the well. The chamber was incubated for 24 hours and the cells, which invaded the Matrigel membrane and migrated to the lower surface of the membrane, were counted. These data demonstrate an inhibitory effect of epigallocatechin gallate and a combination of ascorbic acid, proline and lysine on Matrigel invasion and migration of HCT116 human colon cancer cells.

Example 3

Human melanoma cells A2058 (ATCC) were seeded on a Matrigel insert in a perfected Matrigel invasion chamber (BD). Conditioned media from normal human skin fibroblasts (Clonetics) supplemented with various agents were added to the well. The chamber was incubated for 24 hours and the cells, which invaded the Matrigel membrane and migrated to the lower surface of the membrane, were counted. These data demonstrate the inhibitory effect of epigallocatechin gallate and a combination of ascorbic acid, proline and lysine on Matrigel invasion and migration of human melanoma A2058 cells.

Example 4

In this experiment, human melanoma cells A2058 (ATCC) were seeded on a Matrigel insert in an improved Matrigel invasion chamber (BD) in the presence of:

A: conditioned media from normal human skin fibroblasts (Clonetics) and

B: the same media supplemented with different nutrients as indicated in the legends of Figures 4. After 24 hours of incubation, cells that invaded the Matrigel membrane and migrated to the lower surface of the membrane were examined under a microscope and counted.

These data demonstrate the apoptotic effect of epigallocatechin gallate and the combination of ascorbic acid + proline + lysine on A2058. AND; Human melanoma cells in tissue culture medium. B: Human melanoma cells in tissue culture medium containing ascorbic acid (100 μM), proline (140 μM), lysine (400 μM), and EGCG (20 μg/ml). Note: the cells were destroyed.

Example 5

Investigation of cancer cell proliferation

A2058 melanoma cells

Figure 5 shows the effect of 10, 20 and 50 μg/ml EGCG, without and with the addition of lysine, proline and ascorbic acid on the proliferation of melanoma cells. Neither lysine, nor proline, nor ascorbic acid, nor EGCG at 10 and 20 μg/ml had any significant effect on cell proliferation. However, EGCG at 50 μg/ml significantly reduced the number of cells to 30%. A similar effect was observed with lysine, proline and ascorbic acid.

MDA-MB-231 breast cancer cells

In these experiments, the basal medium was supplemented with 0, 10, 20, 50, 100 or 200 μg/ml EGCG (Figure 6). The results show that supplementing the basal medium with 50, 100 and 200 μg/ml EGCG significantly reduced the number of cells to 66.1 ± 5.3%; 33.6 ± 2 % and 29.6 ± 0.8% in comparison with the respective non-supplemented controls. Concentrations of EGCG in the cell medium up to 20 μg/ml did not have any significant inhibitory effect on cell proliferation.

We also investigated the effects of ascorbic acid, lysine, proline and different concentrations of EGCG on the proliferation of cancer cells. Figure 7 shows insignificantly reduced cell number up to 86.1 + 1.93% with ascorbic acid, lysine and proline. Further addition of 20, 50 and 100 μg EGCG to this combination significantly reduced the number of cells to 74 ± 5.8%; 64.8 ± 1.6% and 22 ± 5% compared to the respective control group.

HCT 116 colon cancer cells

While the inhibitory effect of ascorbic acid, proline and lysine on the proliferation of colon cancer cells was not emphasized, the combination of ascorbic acid, proline and lysine with 20 μg/ml EGCG significantly reduced the number of cells up to 69 ± 0.5% (Figure 8). The higher concentration of EGCG in this combination (50 μg/ml) drastically reduced the number of cells to 4.6 ± 0.3%.

The antiproliferative effect of the nutrient combinations used in these studies varied with the type of cancer cells. In breast cancer cells, the combination of ascorbic acid, proline and lysine with EGCG had greater antiproliferative effects than when these nutrients were used individually. Melanoma and colon cancer cells exposed to the combination of ascorbic acid, proline and lysine had no effect on their proliferation. However, combining these nutrients with 20 μg/ml EGCG resulted in a significant reduction in the number of colon cancer cells, but not melanoma cells. Colon cancer cells seem to be more sensitive than breast cancer cells and melanoma cells to the smallest combination of ascorbic acid, proline, lysine and EGCG. Proliferation of colon cancer cells was almost completely reduced (4.6%) when ascorbic acid, proline and lysine were administered with 50 μg/ml EGCG.

Example 6

Gelatinase zymographic tests

The effect of EGCG on melanoma cells with basal medium and ascorbic acid, proline and lysine supplementation on MMP expression is shown in Figure 9 using gelatinase zymography. Melanoma cells showed two bands corresponding to MMP-2 and MMP-9. The combination of ascorbic acid, lysine and proline has no effect on MMP-band expression compared to the basal medium. However, EGCG inhibited the expression of both MMP-2 and MMP-9 in a dose-dependent manner. The intensity of the stripes in basal conditions and in the combination of ascorbic acid, lysine and proline was the same.

Example 7

Studies of extracellular matrix invasion and migration

The inhibitory effects of the combination of ascorbic acid, proline and lysine used separately, as well as together with different concentrations of EGCG, were tested. The effects of these combinations on the extracellular matrix were investigated using preformed Matrigel matrices, which are routinely used to determine the level of invasive potential of various cancer cell lines.

MDA-MB-231 breast cancer cells

Figure 10 shows the results of invasion into Matrigel of breast cancer cells incubated in the presence of ascorbic acid, proline and lysine. Invasion of cancer cells incubated in a combination of ascorbic acid, proline and lysine was reduced to 48.1 ± 22.1% compared to cells incubated in medium without supplements. In medium supplemented with only 20 μg/ml EGCG, the number of invasive cells decreased to 69.5 ± 27.4%. Complete inhibition of matrix invasion by breast cancer cells was achieved in the presence of higher concentrations of EGCG (50 μg/ml and 100 μg/ml).

In another series of studies, supplementing the medium with 100 μM ascorbic acid reduced invasion by 36%. Supplementation with ascorbic acid and 140 μM proline further reduced invasion by 47%. Using 400 μM lysine with ascorbic acid and proline in the supplement further reduced invasion by 67%; lysine shows a linear response in enhancing the effects of ascorbic acid and proline up to the level of 800 μM.

Figure 10 also shows that the combination of ascorbic acid, proline and lysine as well as 20 μg/ml EGCG was effective in completely stopping the invasion of cancer cells through the extracellular matrix. This combination made it possible to reach the maximum inhibitory effect on the invasion of cancer cells without the need to use high concentrations of individual nutrients. As such, ascorbic acid, proline and lysine together with EGCG allowed to completely stop matrix invasion by breast cancer cells at a lower level of EGCG (20 μg/ml).

HCT116 colon cancer cells

Figure 11 shows that the combination of ascorbic acid, proline and lysine significantly reduced colon cancer cell invasion up to 67.2 ± 3.7%. EGCG used alone at 20 μg/ml reduced invasion up to 44.9 ± 3.3%, while the combination of ascorbic acid, proline and lysine, and 20 μg/ml EGCG had a synergistic effect reducing colon cancer cell invasion up to 24.9 ± 4.6%.

A2058 melanoma cells

Figure 12 shows that the combination of ascorbic acid, proline and lysine was effective in reducing the number of invasive cells up to 88.2 ± 4%, however this decrease was not statistically significant. Combining these nutrients with as little as 20 μg/ml EGCG was effective in reducing the number of invasive cells to zero.

These results show that the use of ascorbic acid, proline and lysine with EGCG allowed us to obtain a drastic reduction in the number of cells that invaded and migrated through the Matrigel membrane at lower levels of EGCG. Invasion was reduced to zero using as low a level as 20 μg/ml EGCG with ascorbic acid, proline and lysine in breast cancer cells and melanoma cells. The beneficial results of this combination were not as spectacular with colon cancer cells as they were with breast cancer cells. The level of EGCG had to be at 50 μg/ml to obtain a 90% reduction in invasion by these cells. In this study, no changes were observed in the expression of MMPs in melanoma cells, although EGCG has an inhibitory effect on their expression in a dose-dependent manner.

These lines of research convincingly demonstrate that the subject nutritional pharmaceutical formulation containing ascorbic acid, proline, lysine and at least one polyphenolic compound exerts effective anti-proliferative and anti-invasive effects on cancer cells.

Example 8

The following nutrient pharmaceutical formulations (Tables 1-5) and EPICAN FORTE™ (Table 6) are illustrated for their preparation. These formulations, which contain ascorbic acid, proline, lysine and at least one polyphenolic compound, have been found to be effective in blocking cancer cell invasion and cancer cell metastasis.

Table 1 Formulation 1

[image] mg = milligrams

mcg = micrograms

"%" refers to the weight % of an individual ingredient in the total weight of the formulation

Table 2 Formulation 2

[image] mg = milligrams

mcg = micrograms

"%" refers to the weight % of an individual ingredient in the total weight of the formulation

Table 3 Formulation 3

[image] mg = milligrams

mcg = micrograms

"%" refers to the weight % of an individual ingredient in the total weight of the formulation

Table 4 Formulation 4

[image] mg = milligrams

mcg = micrograms

"%" refers to the weight % of an individual ingredient in the total weight of the formulation

Table 5 Formulation 5

[image] mg = milligrams

mcg = micrograms

"%" refers to the weight % of an individual ingredient in the total weight of the formulation

Table 6 Formulation for EPICAN FORTE™

Dosage size: 6 capsules (Suggested use: two capsules three times a day, suitable with meals)

[image] mg = milligrams

mcg = micrograms

EPICAN FORTE™ is a U.S. brand name. applications in the process

Other ingredients: Vegetarian capsules (hydroxypropyl methylcellulose), silicon dioxide, cellulose and magnesium stearate.

Example 9

Effects of EPICAN FORTE™ on human cancer cells

The effects of EPICAN FORTE™ on human cancer cells were investigated. Metastatic parameters such as expression of matrix metalloproteinases (MMP) using gelatinase zymography, invasion potential through Matrigel and proliferation/growth using MTT assays were examined. The protocols for these analyzes are detailed as above. Several human cancer cell lines were used: skin cancer - melanoma 2058 cells, liver cancer - HepG2 cells, fibrosarcoma - HT 1080 cells, colon cancer - HCT 116, breast cancer - ER+/-MCF-7 and breast cancer ER-/- MDA-MB-231.

The following tables (Tables 7 and 8) summarize the results:

Effects of EPICAN FORTE™ on the proliferation/growth of human cancer cell lines:

Table 7 Doses treated with EPICAN FORTE™ μg/ml: Percentage of control

[image]

Effects of EPICAN FORTE™ on Matrigel invasion and migration of human cancer cells:

Table 8 Treatment doses with EPICAN FORTE™ μg/ml: Percentage of inhibition

[image]

Effects of EPICAN FORTE™ on the expression of matrix metalloproteinases (MMP) in human cancer cell lines:

Melanoma cells: Melanoma cells showed two bands in gelatinase zymography corresponding to MMP-2 and MMP-9. EPICAN FORTE™ inhibited the expression of MMP-2 and MMP-9 in a dose-dependent manner. The expression of MMP-2 and MMP-9 was significantly inhibited at a concentration of 100 μg/ml of EPICAN FORTE™ and was virtually undetectable at a concentration of 1000 μg/ml.

HepG2 cells: Like melanoma cells, HepG2 also expressed two bands corresponding to MMP-2 and MMP-9. EPICAN FORTE™ inhibited the expression of MMP-2 and MMP-9 at concentrations of 500 and 1000 μg/ml.

Fibrosarcoma HT-1080: HT-1080 cells showed two bands for MMP-2 and MMP-9. EPICAN FORTE™ also inhibited the expression of both bands in a dose-dependent manner. Very faint streaks were seen at concentrations of 500 and 1000 μg/ml.

Colon cancer HCT 116: Colon cancer cells showed only one band in zymography corresponding to MMP-2, which completely disappeared at a concentration of 100 μg/ml.

MCF-7 and MDA-MB-231: These cancer cell lines did not show any MMP bands under our experimental concentration, which were similar to other cancer cell lines.

Invasion of MDA-MB-231 through Matrigel was inhibited by 50%, 60%, and 95% at respective 10, 50, and 100 μg/ml concentrations of EPICAN FORTE™. EPICAN FORTE™ was not toxic to MDA-MB-231 at 10 μg/ml and showed mild toxicity at 100 μg/ml. However, it showed significant toxicity at 1000 μg/ml. Neither MMP-2 nor MMP-9 was expressed by zymography. In contrast, EPICAN FORTE™ was not toxic to MCF-7, even at 500 μg/ml, and showed mild toxicity at 1000 μg/ml. MCF-7 were not invasive and did not express MMP activity.

EPICAN FORTE™ inhibits the expression of MMP-2 and MMP-9 in a dose-dependent manner. The expression of MMP-2 and MMP-9 was significantly inhibited at a concentration of 100 μg/ml EPICAN FORTE™, and in fact could not be detected at a concentration of 10 μg/ml. EPICAN FORTE™ used at concentrations of 10 and 100 μg/ml did not significantly affect cell viability, and at 100 μg/ml it showed cytotoxicity in the range of 10-40 percent, depending on the type of cells. Invasion of melanoma cells, MDA-MB-231 cells, and co-culture of melanoma cells with NHDF through Matrigel was significantly reduced in a dose-dependent manner. Invasion of HT-1080 cells through Matrigel was inhibited by 10%, 50%, 70% and 100% at 10, 100, 200, 1000 μg/ml, respectively. Interestingly, EPICAN FORTE™ was not toxic to HT-1080 cells at 100 μg/ml. These results demonstrate that EPICAN FORTE™ is highly effective for several cancer cell lines and also in co-culture. These observations reveal that EPICAN FORTE™ may provide a natural therapeutic basis that makes it a valuable and promising candidate for the treatment of human cancer.

Example 10

Effects of EPICAN FORTE™ on normal human cell lines

The effects of EPICAN FORTE™ on normal human cells were investigated. Parameters similar to those for cancer cell lines were used; namely zymography for MMP expression, invasion through Matrigel and proliferation/growth with MTT experiments. Several normal human dermal fibroblasts (NHDF) were used, including: human chondrocytes and human stromal cells.

The following tables (Tables 9, 10 and 11) summarize the effects of EPICAN FORTE™ on proliferation/growth, metastatic invasion and MMP production in normal human cells:

Table 9 Effects of EPICAN FORTE™ on proliferation/growth of normal human cells. EPICAN FORTE™ treatment doses (μg/ml): Percentage of control

[image]

Table 9 Effects of EPICAN FORTE™ on invasion of normal human cells into Matrigel. Treatment doses with EPICAN FORTE™ (μg/ml): Percentage of inhibition

[image]

The effects of EPICAN FORTE™ on the expression of matrix metalloproteinases (MMPs) in normal human cells are summarized as follows:

NHDF: NHDF expressed only one band in zymography corresponding to MMP-2, which actually disappears at a concentration of 1000 μg/ml EPICAN FORTE™.

Chondrocytes: Chondrocytes also showed only one band corresponding to MMP-2. EPICAN FORTE™ inhibited MMP-2 expression in a dose-dependent manner. MMP-2 expression was significantly inhibited at a concentration of 100 μg/ml EPICAN FORTE™ and completely disappeared at a concentration of 200 μg/ml EPICAN FORTE™.

Stromal cells: Stromal cells also showed only one band corresponding to MMP-2. EPICAN FORTE™ inhibited MMP-2 expression in a dose-dependent manner. A very faint band was seen at concentrations of 50 and 100 μg/ml, which was not detected at concentrations of 200 and 500 μg/ml.

In summary, the results indicated that EPICAN FORTE™ inhibited MMP2 expression in a dose-dependent manner. MMP-2 expression was significantly inhibited at a concentration of 100 μg/ml EPICAN FORTE™ and was virtually undetectable at a concentration of 200 μg/ml. In addition, it was also found that the invasion of chondrocytes through Matrigel was inhibited by 50%, 85% and 95% at 10 μg/ml, 100 μg/ml and 200 μg/ml. At 500 μg/ml, invasion was completely reduced to 0%.

EPICAN FORTE™ was not toxic to chondrocytes, even at a concentration of 200 μg/ml. In fact, EPICAN FORTE™ exerted a cell proliferation effect, a 70% increase in cell proliferation at 200 μg/ml, 70% more than the control. A mild toxic effect was observed only at 500 μg/ml. These results demonstrate that EPICAN FORTE™ is effective in inhibiting MMP-2 expression and that EPICAN FORTE™ represents a novel anti-inflammatory nutrient formulation as a natural approach to inhibit MMP production and extracellular matrix degradation in osteoarthritis and other related diseases including excessive cartilage degradation. .

Although the in vivo experimental lines of EPICAN FORTE™ have just begun research, two cases have already been reported that prove the value of EPICAN FORTE™ as a cancer treatment medication.

One female patient diagnosed with a brain tumor was treated with chemotherapy and radiotherapy without success and was given up at the end of April 2002. By chance, they started treating that patient with Epican-Forte in the recommended doses. At the end of August 2002, MRTS showed that the tumor had disappeared.

A 64-year-old male patient who had a highly elevated PSA tumor marker level in May 2002 (59.9 μg/l) was diagnosed with prostate cancer metastases along the lymphatic vessels of the aorta. He started treatment with Epican-Forte in the recommended doses. Three months later, the PSA level dropped to 0.9 μg/l. A CT scan at the end of October 2002 showed that previously visible lymphatic metastases were no longer evident, and his prostate was of normal size.

A number of embodiments of the present invention are described. Regardless, it should be understood that various modifications can be made without departing from the spirit and scope of this invention.

Claims (33)

1. Nutritive pharmaceutical composition useful in the treatment of cancer, characterized in that it contains: a) one ascorbic compound; b) one L-lysine compound; c) one L-proline compound; and d) at least one polyphenolic compound selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin and catechin, in which compounds a) to c) enhance the activity of the polyphenol compound in blocking cancer proliferation and metastasis. 2. Nutritive pharmaceutical composition according to claim 1, characterized in that the ascorbic compound is selected from the group consisting of ascorbic acid, pharmaceutically acceptable ascorbate salts, ascorbate esters and/or their mixtures. 3. Nutritional composition according to claim 2, characterized in that the pharmaceutically acceptable ascorbate salt is calcium ascorbate or magnesium ascorbate. 4. Nutritional composition according to claim 2, characterized in that the ascorbate ester is ascorbyl palmitate. 5. Nutritive pharmaceutical composition according to claim 1, characterized in that the lysine compound is selected from the group consisting of lysine hydrochloride and pharmaceutically acceptable lysine salts. 6. Nutritive pharmaceutical composition according to claim 1, characterized in that the proline compound is selected from the group consisting of proline hydrochloride and pharmaceutically acceptable proline salts. 7. Nutritive pharmaceutical composition according to patent claim 1, characterized in that it further contains some trace element, selected from the group consisting of selenium, copper, manganese, calcium and magnesium. 8. Nutritive pharmaceutical composition according to patent claim 1, characterized in that it further contains one amino acid. 9. Nutritive pharmaceutical composition according to claim 8, characterized in that the amino acid is arginine. 10. Nutritive pharmaceutical composition according to claim 1, characterized in that it further contains N-acetyl cysteine. 11. Nutritive pharmaceutical composition useful in the treatment of cancer, characterized in that it contains: a) one ascorbic compound; b) one L-lysine compound; c) one L-proline compound; d) N-acetyl cysteine; and e) at least one polyphenolic compound selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin and catechin, in which compounds a) to d) enhance the activity of the polyphenolic compound in blocking cancer proliferation and metastasis. 12. Nutritive pharmaceutical composition according to patent claim 11, characterized in that the ascorbic compound is selected from the group consisting of ascorbic acid, pharmaceutically acceptable ascorbate salts, ascorbate esters and/or their mixtures. 13. Nutritional composition according to claim 12, characterized in that the pharmaceutically acceptable ascorbate salt is calcium ascorbate or magnesium ascorbate. 14. Nutritional composition according to claim 12, characterized in that the ascorbate ester is ascorbyl palmitate. 15. Nutritive pharmaceutical composition according to patent claim 11, characterized in that the lysine compound is selected from the group consisting of lysine hydrochloride and pharmaceutically acceptable lysine salts. 16. Nutritive pharmaceutical composition according to claim 11, characterized in that the proline compound is selected from the group consisting of proline hydrochloride and pharmaceutically acceptable proline salts. 17. Nutritive pharmaceutical composition according to patent claim 11, characterized in that the composition further contains one trace element selected from the group consisting of selenium, copper, manganese, calcium and magnesium. 18. Nutritive pharmaceutical composition according to patent claim 11, characterized in that it further contains one amino acid. 19. Nutritive pharmaceutical composition according to claim 18, characterized in that the amino acid is arginine. 20. Nutritive pharmaceutical composition useful in the treatment of cancer, characterized in that it contains: ascorbic acid, calcium ascorbate, magnesium ascorbate, ascorbyl palmitate, polyphenols, N-acetyl cysteine, lysine, proline, arginine, selenium, copper and manganese. 21. Nutritive pharmaceutical composition according to patent claim 20, characterized in that the composition contains 250 mg of ascorbic acid, 250 mg of calcium ascorbate, 250 mg of magnesium ascorbate, 250 mg of ascorbyl palmitate, 1000 mg of polyphenols, 200 mg of N-acetyl-cysteine, 1000 mg of lysine, 750 mg proline, 500 mg arginine, 30 mcg selenium, 2 mg copper and 1 mg manganese. 22. Nutritive pharmaceutical composition according to patent claim 20, characterized in that the composition contains 25 mg of ascorbic acid, 25 mg of calcium ascorbate, 25 mg of magnesium ascorbate, 25 mg of ascorbyl palmitate, 200 mg of polyphenols, 10 mg of N-acetyl-cysteine, 50 mg lysine, 25 mg proline, 50 mg arginine, 1 mcg selenium, 20 mcg copper and 50 mcg manganese. 23. Nutritive pharmaceutical composition according to patent claim 20, characterized in that the composition contains 5000 mg of ascorbic acid, 5000 mg of calcium ascorbate, 5000 mg of magnesium ascorbate, 5000 mg of ascorbyl palmitate, 5000 mg of polyphenols, 1500 mg of N-acetyl-cysteine, 5000 mg lysine, 3000 mg proline, 3000 mg arginine, 200 mcg selenium, 9 mg copper and 10 mg manganese. 24. Nutritive pharmaceutical composition according to patent claim 20, characterized in that the composition contains 250 mg of ascorbic acid, 250 mg of calcium ascorbate, 250 mg of magnesium ascorbate, 250 mg of ascorbyl palmitate, 1000 mg of polyphenols, 200 mg of N-acetyl-cysteine, 1000 mg of lysine, 750 mg proline, 500 mg arginine, 100 mcg selenium, 2 mg copper, 1 mg manganese, 500 mg calcium and 400 mg magnesium. 25. Nutritive pharmaceutical composition according to patent claim 20, characterized in that the composition contains 250 mg of ascorbic acid, 250 mg of calcium ascorbate, 250 mg of magnesium ascorbate, 250 mg of ascorbyl palmitate, 1000 mg of polyphenols, 200 mg of N-acetyl-cysteine, 1000 mg of lysine, 750 mg proline, 500 mg arginine, 100 mcg selenium, 2 mg copper, 1 mg manganese, 500 mg calcium, 400 mg magnesium and 200 mg citrus bioflavonoids. 26. Nutritive pharmaceutical composition useful in the treatment of cancer, characterized in that it contains: L-lysine, L-proline, L-arginine, ascorbic acid, calcium, magnesium, polyphenols, N-acetyl-cysteine, selenium, copper and manganese. 27. Nutritious pharmaceutical composition according to patent claim 26, characterized in that the composition contains 1000 mg of L-lysine, 750 mg of L-proline, 500 mg of L-arginine, 710 mg of ascorbic acid, 22 mg of calcium, 50 mg of magnesium, 1000 mg of polyphenols , 200 mg of N-acetyl-cysteine, 30 mcg of selenium, 2 mg of copper and 1 mg of manganese. 28. Nutritive pharmaceutical composition according to patent claim 27, characterized in that ascorbic acid is selected from the group consisting of ascorbic acid, calcium ascorbate, magnesium ascorbate and ascorbyl palmitate. 29. Nutritive pharmaceutical composition according to patent claim 27, characterized in that the polyphenol is selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, catechin and other pharmaceutically acceptable polyphenolic salts and/or their mixtures. 30. A method for treating cancer in an individual, characterized by the fact that it contains the step of administering to that individual a nutritional pharmaceutical composition according to any of claims 1-29. 31. The method according to patent claim 30, characterized in that the cancer is selected from the group consisting of melanoma cancer, breast cancer, colon cancer, lung cancer and brain cancer. 32. A method for the treatment of an inflammatory disease in an individual, indicated by the fact that it contains the step of administering to that individual a nutritional pharmaceutical composition according to any of patent claims 1-29. 33. The method according to claim 32, characterized in that the inflammatory disease is osteoarthritis.